HEScGRO

Animal Component-Free Medium for Human Embryonic Stem Cell Research

HEScGRO Medium is the first commercially available, animal component-free (ACF) medium that has been successfully tested for human embryonic stem cell culture. No other hES cell medium is available as a serum-free, ready-to-use and complete formulation. HEScGRO has been extensively tested on licensed hES cell lines and has been shown to maintain hES cells in their undifferentiated state.

Millipore’s hES cell medium is designed to support the growth and expansion of undifferentiated human ES cells and is specially formulated to meet the special requirements of human embryonic stem cell culture. It has been successfully tested and proven to maintain the pluripotential nature of several hES cell lines including: MEL-1, MEL-2 and H1. This medium is defined, serum-free and animal component- free, and does not require additional supplementation to maintain cells in their pluripotent state.

Animal Component Free formulation
Tested on licensed cell lines including MEL-1, MEL-2 and H1 Cells retain pluripotent characteristics after multiple passaging
Complete Medium is prefiltered; no supplementation required
Supplied as 5 x 100 mL aliquots – Convenient, maximizes shelf life

Animal-Component Free Formulation

Human embryonic stem cells have enormous therapeutic potential and provide a useful system for studies of development. To close the gap between research use and clinical therapy, an animal-free model system is required. Until now, the only viable option for human embryonic stem cell culture has involved media containing animal products, such as serum, and animal-derived growth factors and reagents. These animal derived components, such as FBS, are undefined, subject to wide variability and may contain factors which promote differentiation of hES cells. These inherently indefinable animal components may also contain toxic proteins or immunogens and adventitious animal pathogenic contamination which may introduce uncertainty for immunological or developmental studies.

Tested with Qualified hES Cell Lines

HEScGRO ACF medium has been extensively tested with licensed hES cell lines: MEL-1, MEL-2 and H1. Data demonstrates that these cell lines can be successfully passaged multiple times without significant differentiation, confirmed by morphology, marker expression patterns and karyotypic data. Cells cultured with HEScGRO medium have also been shown to readily differentiate into a variety of cell lineages.

HEScGRO-Cultured hES Cells Retain Pluripotent Cell Characteristics

Morphology

MEL-cell lines cultured with HEScGRO ACF Medium maintain their undifferentiated state. A typical assay consists of greater than 5 passages; morphology and karyotype have been maintained for at least 20 passage

Marker Expression Patterns

MEL-1 and MEL-2 cells expressed pluripotent markers after 5 passages of culture with HEScGRO. Alkaline Phosphatase, Oct-4, SSEA-3 marker expression confirmed.

Complete Media – No Supplementation Required

sHEScGRO ACF medium is supplied ready-to-go. There is no need to add bFGF or costly serum- replacement supplements. Recombinant human bFGF is pre-added. Feeder cells are required for best results. Human mitotically inactivated fibroblasts are recommended.

The above figures demonstrate positive marker staining on human MEL-1 ES cell lines following multiple passages of culture with HEScGRO ACF medium. The image on the left shows Oct-4 staining (orange), the middle image shows SSEA-4 staining, and the image on the right shows positive Tra-1-60 staining
.

Morphology of MEL-1 cells at passage 5 following culture with HEScGRO Medium (phase contrast image).

Morphology of H1 cells at passage 5 following culture with HEScGRO Medium (phase contrast image).

Alkaline Phosphatase stained MEL-1 cells at passage 8 following culture with HEScGRO ACF Medium.

MEL-1 and MEL-2

Early Passage Human Embryonic Stem Cells

Early passage cells (p10), maximizing research time
High nuclear/cytoplasmic ratio
Fully characterized with 10 surface markers
Well-defined morphologically
Supplied with MEF cells
Supplied free of intellectual property encumbrances

MEL-1 and MEL-2 are human embryonic stem cell lines that have been approved for stem cell derivation by the Australian National Health and Medical Research Council. They are provided at early passage, which is ideal for maximizing the stable lifespan of the cell line. Late passage cell lines can have a limited workable shelf-life, since karyotype stability may decrease, and pluripotent characteristics (morphology, marker expression) may shift to reflect a multipotent state. The early passage MEL lines provide extended research time in a stable, pluripotent state. The MEL cell lines grow as well defined colonies, with compact cells displaying high nuclear to cytoplasmic ratios and prominent nucleoli. They have been extensively tested with HEScGRO Animal Component-Free Medium and have been shown to maintain complete pluripotency. MEL-1 has a stable XY karyotype, MEL-2 an XX.

Product Specification

	Mel-1 Cells	Mel-2 Cells
Passage No.	P10-P12	P10-P12
Karyotype Analysis	46, XY [p5 and p21]	46, XX [p5 and p36]
Source	Source Donated frozen IVF embryos no longer required for infertility treatment and approved for stem cell derivation by the Australian National Health and Medical Research Council Licensing Committee [License #309709]
Derivation Conditions	Derivation Conditions Derived on mouse fetal fibroblast feeders using 20% Fetal Calf Serum in standard hES cell culture medium [DMEM/Non-essential AA(1xl)/, ß-mercaptoethanol 0.1mM/glutamine 2mM]
Microbiology	Negative for mycoplasma, HTLV-1&2, Hepatitis B&C, HIV, Treponema palladium
Morphology	Well defined colonies with compact cells displaying high nuclear to cytoplasmic ratio and prominent nucleoli
Cell surface Markers Oct-4 GCTM2 TG30 [CD9] Podocalyxin CD24 TRA 1-60 TRA1-81 SSEA-1 SSEA-3 SSEA-4	+ + + + + + + - + +	+ + + + + + + - + +
In Vivo Pluripotency	Endoderm, ectoderm and mesoderm observed representative germ layers in SCID/teratomas

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